The invention relates to a method for operating a preferably confocal laser scanning microscope, the laser system comprising at least one solid-state laser or a diode laser.
In confocal laser scanning microscopy, gas lasers are most often used as illumination sources. The gas lasers take up a quite considerable amount of space, not least because of their gas tube. As a result, compact device designs of the gas lasers cannot be implemented. Furthermore, gas lasers have only a short service life, which results in quite considerable operating costs. In addition, gas lasers need intensive cooling during operation and require complicated air or water cooling, which in turn on the one hand entails costs and on the other hand increases the space requirement of such a laser system. Finally, lasers have a high energy consumption and, at the same time, only a low efficiency.
In addition to the conventional gas lasers, there are also already solid-state lasers. Until now, these have hardly been used in confocal laser scanning microscopy, since these always exhibit intensity fluctuations during the measurement process and these in turn distort the measurement result or superimpose interfering patterns on the actual image information. Stable-intensity solid-state lasers require complicated control mechanisms, which at least largely prevent the occurrence of spiking or relaxation fluctuations. In the embodiments known hitherto from practice, controlled solid-state lasers are complicated and expensive. Uncontrolled solid-state lasers have the drawback of a periodic mode concurrence, which always leads to a pulsed intensity behavior. Although adequate long-term stability can be achieved by means of periodic modulation of the pump light source, this then leads, because of the spiking and because of the relaxation oscillations, to a considerable short-term increase in the laser intensity which, in the case of use in confocal laser scanning microscopy, is associated with undesired interference effects in the process which yields the image. This is not acceptable in the case of the use in confocal laser scanning microscopy.
Spiking and relaxation oscillations are phenomena which are characteristic of most solid-state lasers and semiconductor lasers. In these phases, the recovery times for the population inversion of the excited state are substantially longer than the decay time of the laser resonator. These phenomena do not occur in gas lasers, so that when gas lasers are used, this problem has hitherto been paid hardly any attention.
Irrespective of the application in laser scanning microscopes, approaches to suppressing the spiking behavior of solid-state lasers have already been made. For this purpose, a nonlinear absorber has been inserted into the laser resonator to cause high losses at high intensities. Ultimately, this is a passive solution. Furthermore, an external control loop has already been provided, which operates with a detector and a loss modulator within the resonator. This is an active solution. Even small nonlinear loss elements damp the relaxation behavior considerably. As a result, this approach can be used only conditionally, since hitherto there were no good, rapidly acting optical limiters which have a low threshold value and, within a laser resonator, exhibit sufficiently low losses at the desired intensities. The external control, in accordance with which a loss modulator is provided outside the laser resonator, is therefore possible in principle but is in general complicated and expensive in operation. A stable mechanical design of the laser, acoustic insulation and stable current sources help to minimize the disadvantageous effects.
When frequency-doubling laser systems are used, active stabilization is technically extremely complicated, particularly when it is carried out for an external resonator. When quasi phase-adapted materials are used, a single passage through the nonlinear crystal is also possible, but this is inefficient and barely provides the necessary optical powers.
The present invention is therefore based on the object of specifying a method for operating a preferably confocal laser scanning microscope according to which the use of a solid-state laser or of a diode laser is possible and according to which undesired distortions of the image information are effectively avoided.
The above object is achieved by the features of patent claim 1. According to said claim, a generic methodxe2x80x94using a solid-state laser or a diode laserxe2x80x94is characterized in that the scanning procedure or the recording of data is synchronized with the phase of an at least largely continuous emission of intensity from the laser system.
According to the invention, the synchronization of scanning procedure or data recording and a continuous emission of intensity from the laser system or the laser light source is provided. This achieves the situation where the data recording is synchronized with the time window of a quasi continuous emission of intensity from the solid-state laser or a diode laser, as a result of which no intensity fluctuations of the laser system occur during the measurement process. To this extent, distortion of the measurement result is ruled out.
According to the invention, it is now possible, instead of conventional gas lasers, to use solid-state or diode lasers in the confocal laser scanning microscope. Their suitability for confocal laser scanning microscopy depends on the stability of the intensity of the emitted laser radiation. While simple solid-state lasers without stabilization mechanisms typically exhibit interference in the image intensity when an image is recorded in conjunction with a confocal laser scanning microscope, said interference being attributable to spiking and relaxation fluctuations, these phenomena are avoided when data is being recorded from the confocal laser scanning microscope in the manner according to the invention, because the intensity fluctuations of the laser output, which are mostly regular, are compensated for by means of synchronization. This is ultimately achieved by unavoidable interference, as is generally unavoidable when conventional intensity control systems are used, specifically occurring at a defined point in time. With the knowledge of this situation the time window for the actual recording of the data beginsxe2x80x94intentionally or in a controlled mannerxe2x80x94only after the outlined interfering phase.
In concrete terms, the laser system, comprising a solid-state laser or a diode laser, and the data recording system are synchronized by a control unit, so that the data is recorded in the phase of the at least largely continuous emission of intensity from the laser system. With regard to the synchronization to be carried out, there are in principle two possibilities.
On the one hand, the natural oscillations of the laser system could be adjusted in such a way that the peak behavior of the laser can be used as a trigger pulse or as a synchronization pulse for the data recording system. By utilizing the preferably adjustable natural oscillations of the laser system, its peak behavior supplies a synchronization pulse for the data recording. The synchronization pulse could be fed indirectly or directly from the laser system to the data recording system. In concrete terms, the laser light source or the laser could have a synchronization output, which is connected via a line to the control unit. In this case, the laser could be adjusted such that a periodically repeating laser output intensity waveform occurs. The peak behavior or the spike of the laser is in this case used for synchronization, to be specific preferably via the control unit provided there, as a result of which the recording of the data from the laser scanning microscope is triggered.
On the other hand, the synchronization may be implemented by the laser system used being deliberately influenced by the control unit. To this extent, the laser system receives, via the control unit, a synchronization pulse whereupon, after a dead time, the at least largely continuous state of emission of the laser system is reached for a specific time period, during which the data is then recorded. The synchronization pulse is fed indirectly or directly from the data recording system to the laser system. In concrete terms, the laser system receives the synchronization pulse (trigger) at a time t0, in order that, following a dead time t1 which has to be waited for, the quasi continuous state of emission is reached for the time period t2, which can then be used for the data recording. Ultimately, the control mechanism implemented here is used to stabilize the intensity of the laser system.
In addition, it is advantageous if the laser system sends a control message to the control unit, as a result of which the presence of the at least largely continuous emission of intensity from the laser system is indicated. When this indication occurs, fault-free data recording may be performed.
In principle, it is possible for the recording of each individual pixel in an image to be recorded to be synchronized or triggered. However, the synchronization effort required for this is considerable, because of the plethora of synchronization steps.
It is likewise conceivable for the recording of each individual line of an image to be recorded to be synchronized or triggered. In accordance with the number of pixels to be recorded in total, and the duration of the largely continuous intensity emission phase of the laser system which is suitable for image recording, it is also possible to synchronize or to trigger the recording within the range of a preferably selectable area of an image. If the phase of the quasi continuous emission of intensity from the laser system is adequate, this could also be used to record an entire image, so that the recording of the entire image is possible with a single synchronization. If more time is available, or if the image is composed of a lower number of image points, then the recording of a number of images can be initiated by a single synchronization pulse, so that the recording of a number of imagesxe2x80x94jointlyxe2x80x94is synchronized or triggered. This ultimately depends on the number of image points to be recorded, and on the duration of the largely continuous intensity emission phase of the laser system.
In addition, it is conceivable for the laser system to have frequency-doubling characteristics. The laser system could also operate with an optical parametric oscillator (OPO) and could be used to generate at least two different wavelengths. This leads to quite considerable flexibility of the claimed method.
There are, then, various options of configuring and developing the teaching of the present invention in an advantageous way. For this purpose, reference should be made on the one hand to the claims following patent claim 1, on the other hand to the following explanation of an exemplary embodiment of the invention, using the drawing. In connection with the explanation of the preferred exemplary embodiment of the invention, using the drawing, preferred configurations and developments of the teaching are also explained in general terms. In the drawing: